An enzyme reaction system which contains L-Arg、NADPH、FAD、FMN、BH₄、Ca²⁺ etc,has been studied to determine the activity of nitric oxide synthase (NOS) in rats'brain. Alter being incubated at 37℃ for 80 min in dark with samples,the sulfanilic acid and N-(1-naphthyl)-ethylenediammonium was added to the reaction system to measure the concentration of produced nitrite in system.It was found that the optimum pH for the enzyme reaction was 7.4,Km was 0.1 mmol/L.There was a relationship between the amounts of sample and nitrite concentration in the system. This method is simple and convenient. Intra-assay CV value is 3.69% and inter-assay CV value is 5.16%. The specific activity of nitric oxide synthase in ten rats' brains is (39.61±7.64)nmol/(min·g).